The present invention generally relates to the field of genetic engineering and more particularly to growth factors for endothelial cells and growth factor genes.
Developmental growth, the remodelling and regeneration of adult tissues, as well as solid tumor growth, can only occur when accompanied by blood vessel formation. Angioblasts and hematopoietic precursor cells differentiate from the mesoderm and form the blood islands of the yolk sac and the primary vascular system of the embryo. The development of blood vessels from these early (in situ) differentiating endothelial cells is termed vasculogenesis. Major embryonic blood vessels are believed to arise via vasculogenesis, whereas the formation of the rest of the vascular tree is thought to occur as a result of vascular sprouting from pre-existing vessels, a process called angiogenesis, Risau, et al., Devel. Biol., 125:441-450 (1988).
Endothelial cells give rise to several types of functionally and morphologically distinct vessels. When organs differentiate and begin to perform their specific functions, the phenotypic heterogeneity of endothelial cells increases. Upon angiogenic stimulation, endothelial cells may re-enter the cell cycle, migrate, withdraw from the cell cycle and subsequently differentiate again to form new vessels that are functionally adapted to their tissue environment. Endothelial cells undergoing angiogenesis degrade the underlying basement membrane and migrate, forming capillary sprouts that project into the perivascular stroma. Ausprunk, et al., Microvasc. Rev., 14:51-65 (1977). Angiogenesis during tissue development and regeneration depends on the tightly controlled processes of endothelial cell proliferation, migration, differentiation, and survival. Dysfunction of the endothelial cell regulatory system is a key feature of many diseases. Most significantly, tumor growth and metastasis have been shown to be angiogenesis dependent. Folkman, et al., J. Biol. Chem., 267:10931-10934 (1992).
Key signals regulating cell growth and differentiation are mediated by polypeptide growth factors and their transmembrane receptors, many of which are tyrosine kinases. Autophosphorylated peptides within the tyrosine kinase insert and carboxyl-terminal sequences of activated receptors are commonly recognized by kinase substrates involved in signal transduction for the readjustment of gene expression in responding cells. Several families of receptor tyrosine kinases have been characterized. Van der Geer, et al., Ann. Rev. Cell Biol., 10:251-337 (1994). The major growth factors and receptors transducing angiogenic stimuli are schematically shown in FIG. 1.
Fibroblast growth factors are also known to be involved in the regulation of angiogenesis. They have been shown to be mitogenic and chemotactic for cultured endothelial cells. Fibroblast growth factors also stimulate the production of proteases, such as collagenases and plasminogen activators, and induce tube formation by endothelial cells. Saksela, et al., Ann. Rev. Cell Biol., 4:93-126 (1988). There are two general classes of fibroblast growth factors, FGF-1 and FGF-2, both of which lack conventional signal peptides. Both types have an affinity for heparin and FGF-2 is bound to heparin sulfate proteoglycans in the subendothelial extracellular matrix from which it may be released after injury. Heparin potentiates the stimulation of endothelial cell proliferation by angiogenic FGFs, both by protecting against denaturation and degradation and dimerizing the FGFs. Cultured endothelial cells express the FGF-1 receptor but no significant levels of other high-affinity fibroblast growth factor receptors.
Among other ligands for receptor tyrosine kinases, the platelet derived growth factor, PDGF-BB, has been shown to be weakly angiogenic in the chick chorioallantoic membrane. Risau, et al., Growth Factors, 7:261-266 (1992). Transforming growth factor xcex1 (TGFxcex1) is an angiogenic factor secreted by several tumor cell types and by macrophages. Hepatocyte growth factor (HGF), the ligand of the c-met proto-oncogene-encoded receptor, also is strongly angiogenic.
Recent evidence shows that there are endothelial cell specific growth factors and receptors that may be primarily responsible for the stimulation of endothelial cell growth, differentiation and certain differentiated functions. The best studied of these is vascular endothelial growth factor (VEGF), a member of the PDGF family. Vascular endothelial growth factor is a dimeric glycoprotein of disulfide-linked 23 kDa subunits. Other reported effects of VEGF include the mobilization of intracellular calcium, the induction of plasminogen activator and plasminogen activator inhibitor-1 synthesis, stimulation of hexose transport in endothelial cells, and promotion of monocyte migration in vitro. Four VEGF isoforms, encoded by distinct mRNA splice variants, appear to be equally capable of stimulating mitogenesis in endothelial cells. However, each isoform has a different affinity for cell surface proteoglycans, which behave as low affinity receptors for VEGF. The 121 and 165 amino acid isoforms of VEGF (VEGF121 and VEGF165) are secreted in a soluble form, whereas the isoforms of 189 and 206 amino acid residues remain cell surface associated and have a strong affinity for heparin.
VEGF was originally purified from several sources on the basis of its mitogenic activity toward endothelial cells, and also by its ability to induce microvascular permeability, hence it is also called vascular permeability factor (VPF). VEGF produces signals through two receptor tyrosine kinases, VEGFR-1 (FLT-1) and VEGFR-2 (KDR/Flk-1), which are expressed specifically on endothelial cells. The VEGF-related placenta growth factor (PlGF) was recently shown to bind to VEGFR-1 with high affinity. PlGF was able to enhance the growth factor activity of VEGF, but it did not stimulate endothelial cells on its own. Naturally occurring VEGF/PlGF heterodimers were nearly as potent mitogens as VEGF homodimers for endothelial cells.
The pattern of VEGF expression suggests its involvement in the development and maintenance of the normal vascular system and in tumor angiogenesis. During murine development, the entire 7.5 day post-coital (p.c.) endoderm expresses VEGF and the ventricular neuroectoderm produces VEGF at the capillary ingrowth stage. See Breier, et al., Development, 114:521-523 (1992). On day two of quail development, the vascularized area of the yolk sac as well as the whole embryo show expression of VEGF. In addition, epithelial cells next to fenestrated endothelia in adult mice show persistent VEGF expression, suggesting a role in the maintenance of this specific endothelial phenotype and function.
Two high affinity receptors for VEGF have been characterized. These are VEGFR-1/Flt-1 (fms-like tyrosine kinase-1) and VEGFR-2/Kdr/Flk-1 (kinase insert domain containing receptor/fetal liver kinase-1). Those receptors are classified in the PDGF-receptor family, but they have seven rather than five immunoglobulin-like loops in their extracellular domain and they possess a longer kinase insert than normally observed in this family. The expression of VEGF receptors occurs mainly in vascular endothelial cells, although some may be present on monocytes and melanoma cells. Only endothelial cells have been reported to proliferate in response to VEGF, and endothelial cells from different sources show different responses. Thus, the signals mediated through VEGFR-1 and VEGFR-2 appear to be cell type specific.
The Flt4 receptor tyrosine kinase (VEGFR-3) is closely related in structure to the products of the VEGFR-1 and VEGFR-2 genes. Despite this similarity, the mature form of Flt4 differs from the VEGF receptors in that it is proteolytically cleaved in the extracellular domain into two disulfide-linked polypeptides. Pajusola et al., Cancer Res., 52:5738-5743 (1992). The 4.5 and 5.8 kb Flt4 mRNAs encode polypeptides which differ in their C-termini due to the use of alternative 3xe2x80x2 exons. The VEGFs do not show specific binding to Flt4 or induce its autophosphorylation.
Expression of Flt4 appears to be more restricted than expression of VEGFR-1 or VEGFR-2. The expression of Flt4 first becomes detectable by in situ hybridization in the angioblasts of head mesenchyme, the cardinal vein, and extraembryonically in the allantois of 8.5 day p.c. mouse embryos. In 12.5 day p.c. embryos the Flt4 signal is observed in developing venous and presumptive lymphatic endothelia, but arterial endothelia appear negative. During later stages of development, Flt4 mRNA becomes restricted to developing lymphatic vessels. Only the lymphatic endothelia and some high endothelial venules express Flt4 mRNA in adult human tissues and increased expression occurs in lymphatic sinuses in metastatic lymph nodes and in lymphangioma. These results support the theory of the venous origin of lymphatic vessels.
Five endothelial cell specific receptor tyrosine kinases, Flt-1 (VEGFR-1), KDR/Flk-1 (VEGFR-2), Flt4, Tie and Tek/Tie-2 have so far been described, which possess the intrinsic tyrosine kinase activity essential for signal transduction. Targeted mutations inactivating Flt-1, Flk-1, Tie and Tek in mouse embryos have indicated their essential and specific roles in vasculogenesis and angiogenesis at the molecular level. VEGFR-1 and VEGFR-2 bind VEGF with high affinity (Kd 16 pM and 760 pM, respectively) and VEGFR-1 also binds the related placenta growth factor (PlGF; Kd about 200 pM), while the ligands for Tie, Tek, and Flt4 have not heretofore been reported.
The present invention provides a ligand for the Flt4 receptor tyrosine kinase. Thus, the invention provides a purified and isolated polypeptide which specifically binds to the Flt4 receptor tyrosine kinase. In a preferred embodiment, the ligand comprises a fragment of the amino acid sequence shown in SEQ ID NO: 33 which specifically binds to the human Flt4 receptor tyrosine kinase.
The present invention also provides one or more precursors of an Flt4 ligand, wherein one such precursor (designated xe2x80x9cprepro-VEGF-Cxe2x80x9d) comprises the complete amino acid sequence (amino acid residues xe2x88x92102 to 317) shown in SEQ ID NO: 33. Thus, the invention includes a purified and isolated polypeptide having the amino acid sequence of residues xe2x88x92102 to 317 shown in SEQ ID NO: 33.
A putative 102 amino acid leader (prepro) peptide has been identified in the amino acid sequence shown in SEQ ID NO: 33. Thus, in a related aspect, the invention includes a purified and isolated polypeptide having the amino acids sequence of residues 1-317 shown in SEQ ID NO: 33.
The expressed Flt4 ligand precursor is proteolytically cleaved upon expression to produce an approximately 23 kD peptide which is the Flt4 ligand (herein designated VEGF-C). Thus, in a preferred embodiment of the invention, an Flt4 ligand is provided which is the cleavage product of the precursor peptide shown in SEQ ID NO: 33 and which has a molecular weight of approximately 23 kD under reducing conditions. A putative VEGF-C precursor, having an observed molecular weight of about 32 kD, also is considered an aspect of the invention.
From the foregoing, it will be apparent that an aspect of the invention includes a fragment of the purified and isolated polypeptide having the amino acid sequence of residues xe2x88x92102 to 317 shown in SEQ ID NO: 33, the fragment being capable of specifically binding to Flt4 receptor tyrosine kinase. In a preferred embodiment, the invention includes a fragment having an apparent molecular weight of approximately 23 kD as assessed by SDS-PAGE under reducing conditions.
In a related aspect, the invention includes a purified and isolated polypeptide capable of specifically binding to Flt4 receptor tyrosine kinase, the polypeptide comprising a fragment of the purified and isolated polypeptide having the amino acid sequence of residues xe2x88x92102 to 317 shown in SEQ ID NO: 33, the fragment being capable of specifically binding to Flt4 receptor tyrosine kinase. Similarly, the invention includes a polypeptide having an amino acid sequence comprising a portion of SEQ ID NO:2, the portion encoding a fragment capable of specifically binding to Flt4.
Evidence suggests that the amino acids essential for retaining Flt4 ligand activity are contained within approximately amino acids 1-115 of SEQ ID NO: 33, and that a carboxy-terminal proteolytic cleavage to produce a mature, naturally-occurring Flt4 ligand occurs within approximately amino acids 115-180 of SEQ ID NO: 33. Accordingly, preferred polypeptides of the invention include polypeptides comprising amino acids 1-115, 1-116, 1-117, 1-118, 1-119, 1-120, 1-121, 1-122, 1-123, 1-124 . . . 1-178, 1-179, and 1-180 of SEQ ID NO: 33, wherein the polypeptides specifically bind to an Flt4 receptor tyrosine kinase. A preferred Flt4 ligand comprises approximately amino acids 1-115 of SEQ ID NO: 33. Another preferred polypeptide of the invention comprises approximately amino acids 1-180 of SEQ ID NO: 33.
The present invention also provides a cDNA encoding a novel polypeptide, designated VEGF-C, that is structurally homologous to VEGF. VEGF-C is a ligand for the FLT4 receptor tyrosine kinase (VEGFR-3), a receptor tyrosine kinase related to VEGFR-1 and VEGFR-2 that does not bind VEGF. VEGFR-3 is expressed in venous and lymphatic endothelia of fetal tissues and predominantly in lymphatic endothelial of adult tissues. Kaipainen et al., Cancer Res., 54:6571-77 (1994); Kaipainen et al., Proc. Natl. Acad. Sci. USA, 92:3566-70 (1995).
Thus, aspects of the invention include purified and isolated nucleic acids encoding polypeptides and polypeptide fragments of the invention; vectors which comprise nucleic acids of the invention; and host cells transformed or transfected with nucleic acids or vectors of the invention. For example, in a preferred embodiment, the invention includes a purified and isolated nucleic acid (e.g., a DNA or an RNA) encoding an Flt4 ligand precursor. Due to the degeneracy of the genetic code, numerous such coding sequences are possible, each having in common the coding of the amino acid sequence shown in SEQ ID NO: 33. As set forth above, the invention includes polypeptides which comprise a portion of the amino acid sequence shown in SEQ ID NO: 33 and which bind the Flt4 receptor tyrosine kinase (herein designated VEGFR-3); the invention also is intended to include nucleic acids encoding these polypeptides. Ligand precursors according to the invention, when expressed in an appropriate host cell, produce, via cleavage, a peptide which binds specifically to the Flt4 receptor tyrosine kinase (VEGFR-3). The nucleotide sequence shown in SEQ ID NO:32 contains a preferred nucleotide sequence encoding the Flt4 ligand (VEGF-C).
The present invention also provides a cell line which produces an Flt4 ligand. In a preferred embodiment, the ligand may be purified and isolated directly from the cell culture medium. Also provided are vectors comprising a DNA encoding the Flt4 ligand, and host cells comprising the vectors. Preferred vectors of the invention are expression vectors wherein nucleic acids of the invention are operatively connected to appropriate promoters and other control sequences, such that appropriate host cells transformed or transfected with the vectors are capable of expressing the Flt4 ligand. A preferred vector of the invention is plasmid pFLT4-L, having ATCC accession no. 97231.
The invention further includes a method of making polypeptides of the invention. In a preferred method, a nucleic acid or vector of the invention is expressed in a host cell, and a polypeptide of the invention is purified from the host cell or the host cell""s growth medium.
In a related embodiment, the invention includes a method of making a polypeptide capable of specifically binding to Flt4 receptor tyrosine kinase, comprising the steps of: (a) transforming or transfecting a host cell with a nucleic acid of the invention; (b) cultivating the host cell to express the nucleic acid; and (c) purifying a polypeptide capable of specifically binding to Flt4 receptor tyrosine kinase from the host cell or from the host cell""s growth media.
The invention also is intended to include purified and isolated polypeptide ligands of Flt4 produced by methods of the invention.
In another aspect, the invention includes an antibody which is specifically reactive with polypeptides of the invention. Antibodies, both monoclonal and polyclonal, may be made against a ligand of the invention according to standard techniques in the art. Such antibodies may be used in diagnostic applications to monitor angiogenesis, vascularization, lymphatic vessels and their disease states, wound healing, or certain hematopoietic or leukemia cells, or they may be used to block or activate the Flt4 receptor.
Ligands according to the invention may be labeled with a detectable label and used to identify their corresponding receptors in situ. Labeled Flt4 ligand and anti-Flt4 ligand antibodies may be used as imaging agents in the detection of lymphatic vessels, high endothelial venules, and Flt4 receptors expressed in histochemical tissue sections. The ligand or antibody may be covalently or non-covalently coupled to a suitable supermagnetic, paramagnetic, electron dense, echogenic, or radioactive agent for imaging. Other, non-radioactive labels, such as biotin and avidin, may also be used.
The present invention also provides diagnostic and clinical applications for claimed ligands. In a preferred embodiment, Flt4 ligands or precursors are used to accelerate angiogenesis, e.g., during wound healing, or to promote the endothelial functions of lymphatic vessels. A utility for VEGF-C is suggested as an inducer of angiogenesis also in tissue transplantation, in eye diseases, in the formation of collateral vessels around arterial stenoses and into injured tissues after infarction. Ligands may be applied in any suitable manner using an appropriate pharmaceutically-acceptable vehicle, e.g., a pharmaceutically-acceptable diluent, adjuvant, or carrier. Ligands also may be used to quantify future metastatic risk by assaying biopsy material for the presence of active receptors or ligands in a binding assay or kit using detectably-labeled ligand. An Flt4 ligand according to the invention also may be used to promote re-growth or permeability of lymphatic vessels in, for example, organ transplant patients. Ligands according to the invention also may be used to treat or prevent inflammation, edema, aplasia of the lymphatic vessels, lymphatic obstruction, elephantiasis, and Milroy""s disease. Finally, Flt4 ligands may be used to stimulate lymphocyte production and maturation, and to promote or inhibit trafficking of leukocytes between tissues and lymphatic vessels or to affect migration in and out of the thymus.
Inhibitors of the Flt4 ligand may be used to control endothelial cell proliferation and lymphangiomas. For example, such inhibitors may be used to arrest metastatic growth or spread, or to control other aspects of endothelial cell expression and growth. Inhibitors include antibodies, antisense oligonucleotides, and peptides which block the Flt4 receptor, all of which are intended as aspects of the invention.
Polynucleotides of the invention such as those described above, fragments of those polynucleotides, and variants of those polynucleotides with sufficient similarity to the non-coding strand of those polynucleotides to hybridize thereto under stringent conditions all are useful for identifying, purifying, and isolating polynucleotides encoding other (non-human) mammalian forms of VEGF-C. Thus, such polynucleotide fragments and variants are intended as aspects of the invention. Exemplary stringent hybridization conditions are as follows: hybridization at 42xc2x0 C. in 5xc3x97SSC, 20 mM NaPO4, pH 6.8, 50% formamide; and washing at 42xc2x0 C. in 0.2xc3x97SSC. Those skilled in the art understand that it is desirable to vary these conditions empirically based on the length and the GC nucleotide base content of the sequences to be hybridized, and that formulas for determining such variation exist. See, e.g., Sambrook et al., Molecular Cloning: a Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory (1989).
Moreover, purified and isolated polynucleotides encoding other (non-human) mammalian VEGF-C forms also are aspects of the invention, as are the polypeptides encoded thereby, and antibodies that are specifically immunoreactive with the non-human VEGF-C variants. Thus, the invention includes a purified and isolated mammalian VEGF-C polypeptide, and also a purified and isolated polynucleotide encoding such a polypeptide.
In one embodiment, the invention includes a purified and isolated polypeptide having the amino acid sequence of residues 1 to 415 of SEQ ID NO: 41, which sequence corresponds to a putative mouse VEGF-C precursor. The putative mouse VEGF-C precursor is believed to be processed into a mature mouse VEGF-C in a manner analogous to the processing of the human prepro-polypeptide. Thus, in a related aspect, the invention includes a purified and isolated polypeptide capable of specifically binding to an Flt4 receptor tyrosine kinase (e.g., a human or mouse Flt-4 receptor tyrosine kinase), the polypeptide comprising a fragment of the purified and isolated polypeptide having the amino acid sequence of residues 1 to 415 of SEQ ID NO: 41, the fragment being capable of specifically binding to the Flt4 receptor tyrosine kinase. The invention further includes purified and isolated nucleic acids encoding the foregoing polypeptides, such as a nucleic acid comprising all or a portion of the sequence shown in SEQ ID NO: 40.